Published online before print November 9, 2006, doi: 10.1161/01.ATV.0000252126.48375.d5
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© 2007 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Establishment of a High Sensitivity Plasma Assay for Human Pentraxin3 as a Marker for Unstable Angina Pectoris
From the Laboratory for Systems Biology and Medicine (K.I., P.C.R., T.H., T.K.), Research Center for Advanced Science and Technology, The University of Tokyo, Japan; the Department of Cardiology (K.I.), Juntendo University Nerima Hospital, Tokyo, Japan; the Perseus Proteomics Inc (A.S., P.C.R., Y.I., M.S., K.M., H.S., I.K.), Tokyo, Japan; the Department of Cardiology (K.M., T.K., H.O., H.D.), Juntendo University School of Medicine, Tokyo, Japan; the Shin Yokohama Cardiovascular Clinic (S.M.), Kanagawa, Japan; and the Istituto Clinico Humanitas (A.M.), Italy.
Correspondence to Tatsuhiko Kodama, Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1, Komaba, Meguro, Tokyo, 153-8904, Japan. E-mail kodama{at}lsbm.org
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Abstract |
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Objective— Plasma pentraxin 3 (PTX3) levels are increased in patients with acute myocardial infarction, yet its involvement in unstable angina pectoris (UAP) remains unclear. To critically evaluate the role of PTX3 in UAP, a sensitive and precise measurement of PTX3 concentration is needed.
Methods and Results— We established a high sensitive plasma ELISA assay system for the detection of PTX3 using monoclonal antibodies. The lower limit of detection of our ELISA was 0.1 ng/mL, sensitivity far greater than the current commercially available kit. Plasma samples were obtained from 162 consecutive patients treated for hypertension, hyperlipidemia, diabetes mellitus, or cardiovascular disease at a physician’s office. PTX3 was not associated with any known coronary risk factors. Additionally, we collected plasma samples from 252 consecutive subjects admitted to a university hospital for coronary artery assessment by coronary angiography. PTX3 was significantly increased in patients in whom coronary intervention was performed. We further analyzed the plasma level of PTX3 in 52 patients with effort angina (EAP) and 16 patients with UAP. Compared with the control group, PTX3 were significantly higher in the UAP group.
Conclusions— The levels of plasma PTX3 were increased in patients with arterial inflammation, especially UAP. This PTX3 detection system will be useful for the prediction of UAP.
Pentraxin 3 (PTX3) are increased in patients with acute myocardial infarction, yet its involvement in unstable angina (UAP) remains unclear. We developed a high sensitivity ELISA system for the detection of PTX3. PTX3 were increased in patients with UAP. This ELISA system will be useful for the prediction of UAP.
Key Words: PTX3 • hs-CRP • atherosclerosis • inflammation • acute coronary syndrome
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Introduction |
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Inflammatory mediators are intimately associated with the cascade of events leading to atherosclerotic plaque initiation, development, and rupture.1 This recognition has led to the evaluation of several markers of inflammation as potential tools for cardiovascular risk prediction. Among the most actively studied biomarkers is high sensitivity C-reactive protein (hs-CRP). The association between moderately elevated hs-CRP levels and an increased risk for development of cardiovascular events is well established.2
CRP and serum amyloid P component (SAP) are well known members of the pentraxin family of proteins. Pentraxins are conserved in evolution from Limulus Polyphemus to man. CRP and SAP are referred to as classical short pentraxins and are acute phase proteins produced in the liver in response to inflammatory mediators. Measurement of CRP is routinely used for diagnosis and monitoring of diverse inflammatory and infectious diseases, whereas SAP is a useful probe to identify amyloid deposits.3–6 Their main inducer is interleukin (IL)-6. PTX3 is the first identified long pentraxin, consisting of a C-terminal pentraxin module coupled with an unrelated N-terminal domain.7,8 PTX3 is structurally related but distinct from classic pentraxins. It differs from the classic short pentraxins in terms of gene organization and localization, ligand recognition, producing cells, and inducing signals.
PTX3 is made in response to primary proinflammatory signals (bacterial products, IL-1, and tumor necrosis factor [TNF], but not IL-6) by diverse cell types, predominantly macrophages and vascular endothelial cells,9–12 but not liver. Due to the fact that PTX3 is produced from vascular endothelial cells and macrophages, instead of from the liver, PTX3 levels may more directly reflect the inflammatory status of the vasculature. Given the similarities and differences between PTX3 and CRP, it is important to assess the usefulness of PTX3 as a novel diagnostic tool for acute coronary syndrome. An initial study in acute myocardial infarction yielded encouraging results,13 and interestingly, it was also PTX3 that we identified as being suppressed by statin treatment in human umbilical vein endothelial cells by cDNA microarray analysis.14
In the present study we report the development of a high sensitivity ELISA system for plasma detection of human PTX3 using two different monoclonal antibodies. Using this system we investigated plasma levels of PTX3 as a role of predictive biomarker in patients with different severities of atherosclerotic disease.
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Reagents
Buffers and solutions used in the ELISA were as follows: PTX3 recombinant protein for calibration curve; immobilization of antibody on plates buffer of 150 mmol/L NaCl saline; blocking buffer of 1% casein in 10 mmol/L phosphate buffer saline (PBS), pH7.4; calibrator dilution buffer of PBS containing 2% bovine serum albumin, 0.2% MICROSIDE III (Anresco Laboratories); sample dilution buffer of PBS, 0.2% MICROSIDE III; enzyme conjugated antibody dilution buffer of Gardian (Pierce Biotechnology Inc) containing 0.1% casein TBS (Pierce Biotechnology Inc), 0.2% MICROSIDE III; washing solution of 0.05% Tween-20 in PBS; substrate solution of TMB (ScyTeK Laboratories Inc); and stop solution (ScyTeK Laboratories Inc). Commercial PTX3 detection system; anti human PTX3 rat monoclonal antibody; MNB4 (ALEXIS Corporation), biotin conjugated anti Human PTX3 rabbit polyclonal antibody (ALEXIS Corporation).
Antigens
Recombinant human PTX3 was expressed in Chinese hamster ovary (CHO) cells as either his-tagged recombinant PTX3 (rPTX3) or as native non-tagged PTX3 and subsequently purified by means of HiTrap chelating Nickel column (GE Healthcare Bio-Science Corp) or ion exchange HiPrep 16/10 Q XL Column (GE Healthcare Bio-Science Corp), respectively, as previously described.10 Polymerization of the purified PTX3 recombinant protein was analyzed by 3% to 8% Tris-Acetate gradient gel SDS-PAGE and SYPRO Ruby Protein Gel Stain (Invitrogen Corporation).
Monoclonal Antibodies
PTX3-deficient mice15 were immunized with intraperitoneal injections of PTX3 in Freund adjuvant (DIFCO, BD) over a period of 4 weeks at 2-week intervals. Fusion of spleen cells from immunized mouse with mouse myeloma cells, NS-1, was performed in a ratio of 10:1 as previously described16. Antibody screening was performed by direct enzyme linked immunosorbent assay (ELISA) method. Monoclonal antibodies were purified from ascites using a HiTrap Protein G HP Column (GE Healthcare Bio-Science Corp). Isotype determination was carried out using an ImmunoPure Monoclonal Antibody Isotyping kit II (Pierce Biotechnology Inc) according to manufacturer’s instructions.
Preparation of Antibody Coated Plates
Monoclonal antibody PPMX0104 was digested by pepsin into F(ab')2 as previously described17. The digested PPMX0104 (100 µL of a 5 µg/mL solution in saline) was coated on a microtiter plate, MaxiSope (Nalge Nunc) at 4°C overnight. After aspirating and washing with washing buffer 5 times, the wells were then blocked with 1% casein in PBS, pH7.4 at 4°C overnight. The plate was aspirated and washed again and the wells were then coated with 150 µL/well of Immunoassay Stabilizer (Advanced Biotechnologies Inc) at 4°C overnight. After which the reagent was discarded and the plate was dried in a desiccation chamber overnight.
Preparation of Anti-PTX3 Detection Antibody-Enzyme Conjugates
One monoclonal antibody (PPMX0105) was digested by pepsin into a F(ab')2 as previously described17 and conjugated with horseradish peroxidase (HRP) according to the manufacturer’s instructions (Peroxidase labeling Kit-SH kit, Dojindo Laboratories).
Assay Procedure
In the typical assay procedure, all incubations were performed at room temperature. Sample dilution (100 µL) was added to the wells of the antibody-coated microtiter plates. Each calibrator or plasma samples (10 µL) were added to the wells and incubated for 1 hour with shaking (first reaction). After the wells were aspirated and washed 5 times with washing buffer (400 µL), Fab’-enzyme conjugate (100 µL) were added to the wells and incubated for 1 hour with shaking (second reaction). The wells were aspirated and washed again, and then substrate solution (100 µL) was added to each well. After 30 minutes of incubation (enzyme reaction), stop solution (100 µL) was added, and the absorbance at 450 nm was measured with a microplate reader system (Towa Labo). Experiments were performed in duplicate except where noted otherwise.
Preparation and Sandwich ELISA of Commercially Available PTX3 Detection System
Commercially available sandwich ELISA for PTX3 was purchased from ALEXIS Corporation (ALEXIS Corporation). Preparation of plates and sandwich ELISA were performed according to manufactures instruction.
Clinical Testing: Population A (Collected From Shin-Yokohama Cardiovascular Clinic)
From January 2006 to April 2006, we measured plasma PTX3 levels in 162 consecutive subjects (unbiased consecutive clinic visitors; age range: 32 to 80 years; 105 men and 57 women) who are treated for hypertension, hyperlipidemia, diabetes mellitus, or cardiovascular disease or come to the clinic for the purpose of an annual health check. We collected baseline data on: body mass index, blood pressure, and history of smoking. We defined prevalent hypertension, diabetes mellitus, or hypercholesterolemia as follows: hypertension, the mean of 2 systolic blood pressure measurements of 
140 mm Hg and/or diastolic pressure 90 mm Hg, or the use of antihypertensive agents; Diabetes mellitus, a hemoglobin A1C level of 5.9%, and/or a history of or current pharmacological treatment for diabetes; Hypercholesterolemia, a total cholesterol level of 220 mg/dL, and/or a history of or current pharmacological treatment for hypercholesterolemia.
Patient Examination
Brachial-Artery Pulse Wave Velocity
All subjects were evaluated for brachial-artery pulse wave velocity (ba PWV). Ba PWV was measured using a volumeplethysmographic apparatus (Form/ABI, Colin Co Ltd). Details of the methodology were as described elsewhere.18,19 The subject was examined while resting in the supine position. Cuffs were wrapped on both brachia and ankles. Pulse volume waveforms at the brachium and ankle were recorded using a semiconductor pressure sensor. Ba PWV was measured after at least 5 minutes rest.
Measuring Carotid Intima–Media Thickness
All patients were evaluated for carotid artery intima–media thickness. Trained technicians used high-resolution B-mode ultrasound to scan three arterial segments bilaterally: the distal 1.0-cm straight portion of the common carotid artery, the carotid bifurcation, and the proximal 1.0 cm of the internal carotid artery.20 Trained readers measured intima–media thickness according to a standardized protocol, and recorded whether or not they saw an apparent plaque. For analysis, we calculated an overall average of the observed mean intima–media carotid thicknesses of the far wall of the six arterial segments.
Population B (Collected From Juntendo University Hospital)
From January 2006 to July 2006, we recruited 406 consecutive subjects, 318 men and 88 women (mean age 65.6±10.5 years, range 21 to 88) admitted to Juntendo university hospital for assessment of ischemic heart disease by coronary angiography (CAG). We excluded patients who have a chronic inflammation status (arteriosclerotic obliterans, aortic aneurysm,21 or rheumatoid arthritis22) because a level of CRP should be increased. We also excluded patients with heart valvular disease because rheumatic fever causes heart valvular disease. And because it is unclear about PTX3 level in patients with renal insufficiency or malignant tumor, patients with more than 2.5 mg/dL level of creatinine or with malignant disease were excluded. Therefore 250 subjects, 197 men and 53 women (mean age 66.3±9.8 years, range 30 to 87), were analyzed. The patients consisted of 52 patients with EAP and 16 with UAP. Stable angina was defined as typical angina precipitated by exertion or emotional stress associated with electrocardiographic change. UAP was defined as augmenting chest pain with a recent increase in frequency and duration, lasting more than 15 minutes or occurring at rest or during minimal effort. These symptoms were combined with ST-segment depression and any changes in biochemical markers of myocardial necrosis (CPK and troponin T). At the time of examination, no subjects in both populations exhibited any evidence of ongoing systemic or cardiac inflammatory diseases. All subjects were of Japanese nationality, and gave their informed consent. This study was approved by the ethical committee of the institution.
Laboratory Measurements
Plasma total cholesterol, high and low-density lipoprotein cholesterol, fast blood sugar (FBS), hemoglobin A1C, hs-CRP, and plasma PTX3 levels were measured. In each case, 4 mL of blood was drawn in EDTA vacuum containers for PTX3 measurement, and were frozen at –20°C until time of assay. Blood samples were obtained immediately before coronary angiography after an overnight fast. In urgent CAG cases, blood samples were drawn at the beginning of the emergency coronary angiographic examination.
Statistical Analysis
The data were expressed as mean±SD. Differences between two groups were analyzed by unpaired t test. All probability values were two-tailed, and values of less than 0.05 were considered to indicate statistical significance. Coefficient of variation (CV) equals the SD divided by the mean value and represents a statistical measure of the dispersion of data points in a data series around the mean, allowing for the calculation of an intra-assay precision. Recovery refers simply to the % of exogenous PTX3 detected after addition to plasma samples. All confidence intervals were calculated at the 95% level.
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Results |
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Purification of PTX3 and Antibody Generation
Recombinant human PTX3 was expressed in CHO cells as either his-tagged rPTX3 or as native non-tagged PTX3 and subsequently purified by means of HiTrap chelating Nickel column or ion exchange HiPrep 16/10 Q XL Column, respectively. Purified His-tagged PTX3 was found to be a monomer, whereas the native form presented as an oligomer (Figure 1a). Purified antigens were immunized into Balb/c mice, MRL/lpr mice, and PTX3-defficient mice. Antibodies produced from Balb/c, MRL/lpr, and PTX3-deficient mice yielded a mixture of N-terminal and C-terminal targeting antibodies (supplemental data 1). Interestingly, antibodies produced in PTX3-deficient mice proved far superior in ELISA compared with antibodies produced in Balb/c or MRL/lpr mice. Immunization with rPTX3-His in PTX3-deficient mice resulted in 10 distinct clones, of which 7 produced N-terminal antibodies and 3 (termed PPMX0104) produced C-terminal antibodies. Whereas, immunization of non-tagged rPTX3 (oligomer) resulted in 16 clones, of which 10 produced N-terminal antibodies and 6 produced C-terminal antibodies (the best of which was termed PPMX0105).
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Development of a PTX3 Assay System
A PTX3 ELISA based assay system was developed using PPMX0104 as the capture antibody and PPMX0105 as the detection antibody. A representative calibration curve based on PTX3 calibrators of 0.5 to 20 ng/mL is shown in Figure 1b and 1c. The absorbance at 450 nm against the amount of calibrator exhibited a linear relation. Intraassay precision was investigated through the analysis in multiplicate (n=5) of 6 different plasma samples, resulting in a coefficient of variation (CV) 
4.1% at all PTX3 concentrations tested. The mean±SD concentrations measured (and CV) were as follows: sample 1, 15.91±0.13 ng/mL (0.8%); sample 2, 7.17±0.10 (1.3%); sample 3, 9.70±0.30 ng/mL (3.0%); sample 4, 0.95±0.02 (1.6%); sample 5, 0.85±0.03 (4.1%); sample 6, 0.67±0.02 (2.9%) (Table S1 in supplemental data 2). Interassay precision was investigated though the analysis of 3 independently performed experiments resulting in a CV 4.3% at all PTX3 concentrations tested. The mean±SD concentrations measured (and CV) were as follows: sample 1, 15.95±0.66 ng/mL (4.1%); sample 2, 7.21±0.22 (3.1%); sample 3, 9.70±0.42 ng/mL (4.3%); sample 4, 0.95±0.02 (2.2%); sample 5, 0.87±0.03 (4.0%); sample 6, 0.66±0.01 (1.8%) (Table S2 in supplemental data 2).
Analytical Recovery and Assay Linearity
Recovery of exogenously added PTX3 from plasma samples ranged from 89.8% to 108.6% respectively (Table 1). Dilution curves of plasma samples showed good linearity (supplemental data 3).
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Assay Specificity and Cross Reactivity
Hemoglobin (up to 500 mg/dL), bilirubin (up to 20 mg/L), and chyle (up to 2000 U as Formazine) had no effect on the present ELISA. The cross-reactivity with CRP and SAP was <0.7%. In regards to PTX3 stability, PTX3 concentrations in whole blood remained relatively unchanged for 8 hours at 25°C with the use of EDTA plasma.
Comparison to Commercially Available PTX3 Assay Kit
To examine the efficacy of our detection system, we compared our newly developed ELISA system to a commercially available ELISA system (Table 1). Whereas our kit relies on monoclonal antibodies as both the capture and detection antibodies, the commercial ELISA uses a rat monoclonal antibody as the capture antibody and a rabbit polyclonal as the detection antibody. The commercial kit relies on a 3 step assay system to detect PTX3 from EDTA plasma at a range of 0.25 to 12 ng/mL. Our assay system relies on a 2 step system to detect PTX3 from EDTA plasma at a range of 0.1 to 20 ng/mL. Analysis of patient samples using both kits revealed a 0 to 4.3% CV for our assay kit and a 4% to 30% CV for the commercially available kit. In addition, we observed a recovery rate of 97.2% in our assay system compared with an 80% recovery rate in the commercially available kit.
Patients for the Clinical Study
Diagnostic Testing of Clinical Samples, Normal Range of PTX3
Before analysis of plasma PTX3 level in patients with atherosclerosis, we measured PTX3 plasma concentration in 56-healthy volunteers to determine a normal range. Their mean age was 40.1±12.1 years, and % male was 59.2%. PTX3 averaged 1.98 ng/mL (95% CI: 1.68 to 2.28).
Diagnostic Testing of Clinical Samples, Independence of PTX3 From Established Risk Factors
Hs-CRP has been known to vary with many conditions, eg, smoking, obesity, age, and diabetes, and it is associated with the metabolic syndrome, which is strongly linked to a proinflammatory state.23 Therefore we analyzed whether plasma PTX3 levels were affected by another coronary risk factor or not. The clinical characteristics of population A are listed in Table 2. 10.8% of patients were current or former smokers, 28.4% had diabetes mellitus, 30.2% had hyperlipidemia, and 46.9% had hypertension. 38.9% of patients showed a BMI of more than 24.2kg/m2. There were two notable features of population A. First, 80.9% of subjects showed a high value of ba PWV. Yamashina et al have reported a ba PWV cutoff value of 14.0 m/s for screening subjects at risk of developing cardiovascular diseases in the general population.24 Second, all of the patients’ conditions were stable. No one had any symptom of chest discomfort or showed abnormal stress electrocardiograms. In population A, plasma levels of PTX 3 were within the normal range, 2.19 ng/mL (95% CI: 2.02 to 2.32). We classified subjects with ba PWV values of more than 14.0m/sec as a relative high risk group for atherosclerotic disease, and examined their plasma PTX 3 levels. Table 3 shows that PTX3 levels were independent of total cholesterol, HDL cholesterol, hemoglobin A1C, smoking status, gender, or obesity, and additionally did not vary with such conditions.
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Association of PTX3 With Coronary Artery Disease
Next, we analyzed plasma PTX3 levels in patients with coronary artery disease. Population B included 197 men and 53 women (mean age 66.3±9.9) (supplemental data 4). In 15 subjects reporting chest pain, yet without any significant stenosis in their coronary artery or evidence of congestive heart failure, exhibited PTX3 plasma concentrations of 1.91 ng/mL (95% CI: 1.52 to 2.31; hence referred to as Normal group). 59.2% of patients were current or former smokers, 43.2% had diabetes mellitus, 72.0% had hyperlipidemia, and 73.2% had hypertension. 48.8% of patients had a BMI of more than 24.2kg/m2. The average plasma PTX3 level was 2.72 ng/mL (95% CI: 2.49 to 2.94). 139 patients in population B had percutaneous coronary intervention or coronary artery bypass graft (Intervention group) performed. The clinical characteristics of the Intervention group and the Nonintervention group are listed in Table 4. There were no significant differences in BMI, blood pressure, or level of total cholesterol, hs-CRP, or hemoglobin A1C between the two groups. However, HDL levels in the Intervention group were lower than that in the Nonintervention group, whereas, LDL, FBS (fast blood sugar), and plasma PTX 3 in the Intervention group levels were significantly higher than that in the Non Intervention group. The Intervention group consisted of 52 patients with EAP and 16 with UAP. Plasma PTX3 levels were significantly higher in patients with UAP than a Normal group (6.09 ng/mL (95% CI: 4.34 to 7.85), P=0.00003, Figure 2). These results indicated that patients who were eligible for coronary intervention showed higher plasma levels of PTX3. Furthermore, because plasma PTX3 levels were significantly increased in patients with UAP, PTX3 levels must be a good predictive diagnostic tool for UAP.
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Discussion |
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In this report, we prepared highly specific monoclonal antibodies against human PTX3 using PTX3-deficient mice, and developed a new sandwich ELISA system. Our assay system can detect PTX3 from plasma at concentrations as low as 0.1 ng/mL, whereas the lowest detectable concentration of commercially available kits is 0.25 ng/mL. Plasma PTX3 levels in healthy volunteers showed around 1.98 ng/mL. Using this system, we measured plasma PTX3 level in two populations of patients, described as population A and B. Plasma PTX3 levels in population A were around 2.19 ng/mL, whereas levels were around 3.04 ng/mL in patients who were eligible for coronary intervention. In patients with UAP, the level of plasma PTX3 was further elevated, to more than three times (around 6.20 ng/mL) those of normal patients.
These results indicate that PTX3 may be a good predictive marker for acute coronary syndrome (ACS). The incidence and potential severity of an ACS makes timely diagnosis and appropriate treatment essential for minimizing morbidity and mortality. Every year in the United States, 
2.5 million patients are admitted to a hospital with an ACS, two thirds of whom are eventually diagnosed with UAP or non–ST-elevation MI.24 Atherosclerotic lesions, composed primarily of a lipid-rich core and a fibrous cap, develop in virtually all major arteries. Autopsy and intravascular ultrasound (IVUS) studies have confirmed the presence of coronary arteriosclerosis lesions in the majority of asymptomatic individuals order than 20 to 30 years of age.
Why some plaques rupture and others do not is not entirely understood, although plaques that are prone to rupture share certain characteristics. The presence of large, eccentric lipid cores and a large percentage of inflammatory macrophages are common findings in fissured or rupture plaques. The role of inflammatory cells and mediators in the degradation and weakening of the protective fibrous cap has recently been recognized as a critical component in the pathogenesis of ACS. The majority of lesions rupture at the site of greatest mechanical stress, often the shoulder of lesions where the fibrous cap is adjacent to normal intima, which is also often the site of greatest inflammatory activity.
To make a diagnosis of ACS, biochemical markers and the resting ECG are key components for the proper assessment of a patient with a suspected ACS. The biochemical markers of myocardial necrosis, predominately CPK, as well as troponin T, are also essential in the diagnosis and prognosis of patients with ACS. Because cardiac troponins are not detected in the blood of healthy individuals and are cardiac specific, they are sensitive and specific for myocardial necrosis. But because UAP does not have any cardiac necrosis theoretically, neither CPK nor troponin T levels are elevated. Furthermore some studies have suggested that 5 to 10% of patients with chest pain and exhibiting a normal ECG will subsequently be diagnosed with unstable angina.25 Therefore, there exists a need to establish an assay system to predict UAP.
PTX3 is an interesting molecule because it was suppressed by statin mostly in endothelial cells, as revealed by gene chip analysis. And, although PTX 3 is in the same family with CRP, its expression pattern is more tissue specific, especially in light of the fact that it is expressed in cells of atherosclerotic lesions, like EC, smooth muscle cells, macrophages, and most recently, in neutrophils. Additionally, in coronary artery at sites distal from the plaque lesion, PTX3 levels were significantly elevated compared with proximal sites, suggesting that PTX3 originates from the atherosclerotic plaque itself, and reflects active atherosclerosis. Therefore, PTX 3 is recognized as the vascular CRP.
Despite the recent recommendations of the Center for Disease Control and the American Heart Association, critical review of the accumulating evidence now suggests that CRP is a relatively poor predictor that does not contribute usefully in this regard. Because elevated CRP levels are intimately linked to conventional CHD risk factors, it would seemingly be less valuable to clinicians. In our study hs-CRP level did not increase in either the EAP or the UAP group (UAP group: hs CRP, 0.48±0.79 mg/L; EAP group: hs CRP, 0.41±1.07 mg/L; P=0.83).
Understanding of the inflammatory cascade allows the consideration of a number of inflammatory markers as potentially useful predictors of prevalent or incident coronary artery disease (CAD). In 1998, the American Heart Association convened Prevention Conference V to discuss strategies for the measurement of markers of inflammation.26 This conference concluded that the most reliable marker to detect acute coronary syndrome must satisfy following criteria: (1) the ability to standardize the assay; (2) independence from established risk factors; (3) association with CAD incidence; (4) the presence of population norms to guide interpretation of results; (5) ability to improve the overall prediction beyond that of traditional risk factors.
Our analysis suggests that PTX3 represents an exceptional biomarker fitting all of these criteria. (1) The ability to standardize the assay: To establish a high sensitivity ELISA system for PTX3, we used different mouse monoclonal antibodies against human PTX3 for capture and signal. Our system could measure plasma PTX3 more stably and precisely, and in a manner more specific with less background than commercially available kits. (2) Independence from established risk factors: Table 3 clearly shows that PTX3 is an independent factor from established risk factors, unlike CRP. Even the cross-reactivity with CRP or SAP was less than 0.7%. (3) Association with CAD incidence: Even in patients with atherosclerotic lesions receiving treatment (so called inactivated inflammatory status) like in population A, plasma PTX3 levels were within normal ranges. But once coronary arteries became eligible for coronary intervention (so called, activated inflammatory status), plasma PTX3 levels increased. These results suggested that this ELISA system represents an ideal tool to predict ACS. (4) The presence of population norms to guide interpretation of results: From 54-volunteer subjects, we determined a normal range of PTX3 of around 1.98 ng/mL (95% CI: 1.68 to 2.28). (5) Ability to improve the overall prediction beyond that of traditional risk factors: Currently, we are performing long term follow-up studies of PTX3 levels in patients receiving treatment, such as statin or aspirin, needed to fully appreciate the overall predictive value of PTX3.
A potential limitation to the present study warrants consideration. We evaluated the predictable utility of PTX3 in a population of patients exclusively with UAP. But because because of the fact that we collected 251 consecutive subjects who had an indication for coronary angiography, only 16 patients could be diagnosed with unstable angina. In general, unstable angina occurs in 6 of every 10 000 people.25 Larger studies are warranted in light of these results.
In summary, we developed a highly sensitive ELISA system for human PTX3 in plasma using monoclonal antibodies. Both intra- and interassay imprecision values were less than 4.1% and 4.3%, respectively. The lower limit of detection of this ELISA was 0.1 ng/mL. The dilution curves of plasma showed good linearity, and the recovery was 97.2%. These attributes are far superior to those of a commercially available kit. We found that patients who were eligible for coronary intervention exhibited high concentrations of plasma PTX3, especially in patients with UAP, exhibiting PTX3 levels three times higher than the normal range. This ELISA system provides a unique tool to assist physicians in predicting patients with UAP.
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Acknowledgments |
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The authors thank Satoko Hamada, Megumi Chujo, Akiko Sawada, Hiroko Iwanari, Aya Sakamoto, and Keiko Katsumi for technical support.
Sources of Funding
This study was supported by the Program of Fundamental Studies in Health Sciences of the NIBIO, NEDO, and by the Fund for Science and Technology from The Ministry of Education, Culture, Sports, Science and Technology in Japan.
Disclosures
None.
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Footnotes |
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K.I. and A.S. contributed equally to this study.
Original received May 27, 2006; final version accepted September 3, 2006.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233–241.[CrossRef][Medline] [Order article via Infotrieve]
2. Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002; 347: 1557–1565.
3. Pepys MB, Baltz ML. Acute phase proteins with special reference to C-reactive protein and related proteins (pentraxins) and serum amyloid A protein. Adv Immunol. 1983; 34: 141–212.[Medline] [Order article via Infotrieve]
4. Gewurz H, Zhang XH, Lint TF. Structure and function of the pentraxins. Curr Opin Immunol. 1995; 7: 54–64.[CrossRef][Medline] [Order article via Infotrieve]
5. Steel DM, Whitehead AS. The major acute phase reactants: C-reactive protein, serum amyloid P component and serum amyloid A protein. Immunol Today. 1994; 15: 81–88.[CrossRef][Medline] [Order article via Infotrieve]
6. Baumann H, Gauldie J. The acute phase response. Immunol Today. 1994; 15: 74–80.[CrossRef][Medline] [Order article via Infotrieve]
7. Breviario F, d’Aniello EM, Golay J, Peri G, Bottazzi B, Bairoch A, Saccone S, Marzella R, Predazzi V, Rocchi M. Interleukin-1-inducible genes in endothelial cells. Cloning of a new gene related to C-reactive protein and serum amyloid P component. J Biol Chem. 1992; 267: 22190–22197.
8. Lee GW, Lee TH, Vilcek J. TSG-14, a tumor necrosis factor- and IL-1-inducible protein, is a novel member of the pentaxin family of acute phase proteins. J Immunol. 1993; 150: 1804–1812.[Abstract]
9. Lee GW, Goodman AR, Lee TH, Vilcek J. Relationship of TSG-14 protein to the pentraxin family of major acute phase proteins. J Immunol. 1994; 153: 3700–3707.[Abstract]
10. Bottazzi B, Vouret-Craviari V, Bastone A, De Gioia L, Matteucci C, Peri G, Spreafico F, Pausa M, D’Ettorre C, Gianazza E, Tagliabue A, Salmona M, Tedesco F, Introna M, Mantovani A. Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component. J Biol Chem. 1997; 272: 32817–32823.
11. Introna M, Alles VV, Castellano M, Picardi G, De Gioia L, Bottazzai B, Peri G, Breviario F, Salmona M, De Gregorio L, Dragani TA, Srinivasan N, Blundell TL, Hamilton TA, Mantovani A. Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites. Blood. 1996; 87: 1862–1872.
12. Basile A, Sica A, d’Aniello E, Breviario F, Garrido G, Castellano M, Mantovani A, Introna M. Characterization of the promoter for the human long pentraxin PTX3. Role of NF-kappaB in tumor necrosis factor-alpha and interleukin-1beta regulation. J Biol Chem. 1997; 272: 8172–8178.
13. Peri G, Introna M, Corradi D, Iacuitti G, Signorini S, Avanzini F, Pizzetti F, Maggioni AP, Moccetti T, Metra M, Cas LD, Ghezzi P, Sipe JD, Re G, Olivetti G, Mantovani A, Latini R. PTX3, A prototypical long pentraxin, is an early indicator of acute myocardial infarction in humans. Circulation. 2000; 102: 636–641.
14. Morikawa S, Takabe W, Mataki C, Kanke T, Itoh T, Wada Y, Izumi A, Saito Y, Hamakubo T, Kodama T. The effect of statins on mRNA levels of genes related to inflammation, coagulation, and vascular constriction in HUVEC. Human umbilical vein endothelial cells. J Atheroscler Thromb. 2002; 9: 178–183.[Medline] [Order article via Infotrieve]
15. Garlanda C, Hirsch E, Bozza S, Salustri A, De Acetis M, Nota R, Maccagno A, Riva F, Bottazzi B, Peri G, Doni A, Vago L, Botto M, De Santis R, Carminati P, Siracusa G, Altruda F, Vecchi A, Romani L, Mantovani A. Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response. Nature. 2002; 420: 182–186.[CrossRef][Medline] [Order article via Infotrieve]
16. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975; 256: 495–497.[CrossRef][Medline] [Order article via Infotrieve]
17. Parham P. On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice. J Immunol. 1983; 131: 2895–2902.[Abstract]
18. Yamashina A, Tomiyama H, Takeda K, Tsuda H, Arai T, Hirose K, Koji Y, Hori S, Yamamoto Y. Validity, reproducibility, and clinical significance of noninvasive brachial-ankle pulse wave velocity measurement. Hypertens Res. 2002; 25: 359–364.[CrossRef][Medline] [Order article via Infotrieve]
19. Tomiyama H, Yamashina A, Arai T, Hirose K, Koji Y, Chikamori T, Hori S, Yamamoto Y, Doba N, Hinohara S. Influences of age and gender on results of noninvasive brachial-ankle pulse wave velocity measurement–a survey of 12517 subjects. Atherosclerosis. 2003; 166: 303–309.[CrossRef][Medline] [Order article via Infotrieve]
20. Bond M, Barnes R, Riley WA, Wilmoth SK, Chambless LE, Howard G, Owens B. High-resolution B-mode ultrasound scanning methods in the Atherosclerosis Risk in Communities Study (ARIC). The ARIC Study Group. J Neuroimaging. 1991; 1: 68–73.[Medline] [Order article via Infotrieve]
21. Norman P, Spencer CA, Lawrence-Brown MM, Jamrozik B. C-Reactive Protein Levels and the Expansion of Screen-Detected Abdominal Aortic Aneurysms in Men. Circulation. 2004; 110: 862–866.
22. Fazzini F, Peri G, Doni A, Dell’Antonio G, Dal Cin E, Bozzolo E, D’Auria F, Praderio L, Ciboddo G, Sabbadini MG, Manfredi AA, Mantovani A, Querini PR. PTX3 in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation. Arthritis Rheum. 2001; 44: 2841–2850.[CrossRef][Medline] [Order article via Infotrieve]
23. Grundy SM, Hansen B, Smith SC Jr, Cleeman JI, Kahn RA. Clinical management of metabolic syndrome: report of the Am Heart Association/National Heart, Lung, and Blood Institute/Am Diabetes Association conference on scientific issues related to management. Arterioscler Thromb Vasc Biol. 2004; 24: e19–e24.
24. Yamashina A, Tomiyama H, Arai T, Hirose K, Koji Y, Hirayama Y, Yamamoto Y, Hori S. Brachial-ankle pulse wave velocity as a marker of atherosclerotic vascular damage and cardiovascular risk. Hypertens Res. 2003; 26: 615–622.[CrossRef][Medline] [Order article via Infotrieve]
25. Braunwald E. Heart Disease, a Text Book of Cardiovascular Medicine (fourth edition). Elseveir; Philadelphia, Pa; 2004: 1243–1273.
26. Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO 3rd, Criqui M, Fadl YY, Fortmann SP, Hong Y, Myers GL, Rifai N, Smith SC Jr, Taubert K, Tracy RP, Vinicor F. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the Am Heart Association. Circulation. 2003; 107: 499–511.
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